ADP-ribosylation, in which the ADP-ribose moiety of NAD is transferred to a target protein, is catalyzed by a family of bacterial toxins and mammalian enzymes. Some toxin ADP-ribosyltransferases (e.g., cholera toxin, diphtheria toxin) appear to be responsible for the diseases caused by the bacterium. Mammalian cells contain enzymes that catalyze reactions similar to some of the bacterial toxins. Some of the mammalian ADP-ribosyltransferases (ARTs) are located both within the cell and on the cell surface, sometimes, linked through a glycosylphosphatidylinositol anchor (ART1), whereas, others ART5 appear to be secreted. A family of the mammalian transferases has been cloned in the laboratory; they display some structural similarities with amino acid identities in the catalytic site. In human airways, epithelial cells lining the lumen and intraluminal cells participate in the innate immune response. These cells express, on their surfaces, arginine-specific ADP-ribosyltransferases (ART), which transfer ADP-ribose from NAD to proteins. During lung inflammation, activated neutrophils release human neutrophil peptides (HNP), small, cationic peptides characterized by a high arginine content. Previously, we found that ART1 modified arginine-14 of HNP-1, altering its biological activities. Human airways, however, express several ARTs and enzymes that metabolize ADP-ribosylated proteins. We investigated the specificity of different ARTs for modification of HNP-1 and the influence of HNP-1 on the activities of ART1 and ART5. HNP-1 was incubated in the presence of NAD and either ART1, 4, or 5, cholera toxin, a bacterial NAD:arginine ADP-ribosyltransferase, or free ADP-ribose. Only ART1, whether synthesized in E.coli, glycosylphophatidylinositol-anchored on transfected NMU cells or expressed endogenously on C2C12 myotubes modified HNP-1. Free ADP-ribose, a product of ART1- and ART5-catalyzed reactions, did not form a covalent linkage to HNP-1. NAD, but not NADP, was a substrate in the ART1-catalyzed modification. HNP-1 at concentrations higher than 5 micromolar inhibited the activities of both ART1 and ART5. Bronchoalveolar lavage fluid from individuals with two common polymorphic forms of ART1, with different activities toward low molecular weight guanidino compounds, contained ADP-ribosylated HNP-1, consistent with the conclusion that the polymorphism was not affecting the modification. Incubation of ADP-ribosylated HNP-1 with pyrophosphatase plus alkaline phosphatase, enzymes present on the surface of airways, generated ribosyl-HNP-1. This compound was considerably more effective than ADP-ribosyl-HNP-1 or HNP-1 in stimulating IL-8 release from A549 cells. Our data suggest that ADP-ribosylaton of HNP-1 is specific for ART1; other arginine-specific ADP-ribosyltransferases and free ADP-ribose did not modify the defensin. Thus, HNP-1 interacts with ADP-ribosyltransferases expressed in human lung. Post-translational modification and metabolism of modified HNP-1 may regulate its biological activities.